人正肺炎病毒在癌细胞系中的溶瘤活性

ONCOLYTIC ACTIVITY OF HUMAN ORTHOPNEUMOVIRUS IN CANCER CELL LINES.

作者信息

Aziz I M, Bhat R, Farrag M A, Almajhdi F N

机构信息

College of Science, Alfaisal University, Riyadh 11533, Saudi Arabia.

出版信息

Exp Oncol. 2022 Aug;44(2):113-120. doi: 10.32471/exp-oncology.2312-8852.vol-44-no-2.18084.

DOI:10.32471/exp-oncology.2312-8852.vol-44-no-2.18084

PMID:35964639

Abstract

UNLABELLED

Oncolytic virotherapy is an emerging biotherapeutic platform for selectively infecting cancer cells and triggering apoptosis in a number of malignant cells due to robust viral replication. Studies related to the oncolytic activity of human orthopneumovirus (hOPV) are conflicting.

AIM

This study was designed to elucidate the possible role of hOPV in the modulation of cell growth and apoptosis in cancer cell lines including human epidermoid carcinoma (HEp-2), lung epithelial cell line (A549), and breast cancer cell line (MCF-7).

MATERIALS AND METHODS

The oncolytic activity of hOPV on cancer cells was studied in vitro. The virus titers were determined by tissue culture infectious dose (TCID50/mL) in A549 cell. The cytotoxic effect of the virus on HEp-2, A549, and MCF-7 was determined using MTT and trypan blue dye exclusion test assays. hOPV in the infected cells was detected using real-time reverse transcription polymerase chain reaction (rRT-PCR) and indirect immunofluorescence (IIF) assays. The relative expression of apoptosis-related genes (CASP-3, -8, -9, Bax, Bcl-2, Bcl-XL, TP53, P21) during virus infection was estimated using rRT-PCR assay in comparison with the house-keeping gene (GAPDH).

RESULTS

hOPV infection inhibited the growth of HEp-2, A549, and MCF-7 cells in a dose-and time-dependent manner. At a multiplicity of infection (MOI) of 5, hOPV reduced the viability of A549 cells to about 16%, HEp-2 to 22%, and MCF-7 to 28% (p = 0.001), while no significant inhibitory effect was observed when cells were infected at MOI of 1 and 2. hOPV mRNA and antigens were detected in infected HEp-2, A549, and MCF-7 cells by RT-PCR and IIF. Upon hOPV infection, expression of CASP-3, -8, -9, as well as Bax, TP53, and p21 mRNA increased while expression of Bcl-2, Bcl-xL anti-apoptotic genes decreased. In hOPV-infected A549 cells, the fold increase of CASP-8 and CASP-9, Bax, TP53, and P21 expression exceeded significantly compared to that in HEp-2 or MCF-7 cells.

CONCLUSIONS

Our results provide evidence that hOPV could be a potential candidate for oncolytic virotherapy.

摘要

未标记

溶瘤病毒疗法是一种新兴的生物治疗平台，可通过强大的病毒复制选择性感染癌细胞并触发多种恶性细胞凋亡。关于人正肺炎病毒（hOPV）溶瘤活性的研究结果相互矛盾。

目的

本研究旨在阐明hOPV在调节包括人表皮样癌（HEp-2）、肺上皮细胞系（A549）和乳腺癌细胞系（MCF-7）在内的癌细胞系中细胞生长和凋亡方面的可能作用。

材料与方法

在体外研究hOPV对癌细胞的溶瘤活性。通过在A549细胞中进行组织培养感染剂量（TCID50/mL）测定病毒滴度。使用MTT和台盼蓝染料排除试验测定病毒对HEp-2、A549和MCF-7的细胞毒性作用。使用实时逆转录聚合酶链反应（rRT-PCR）和间接免疫荧光（IIF）试验检测感染细胞中的hOPV。与管家基因（GAPDH）相比，使用rRT-PCR试验估计病毒感染期间凋亡相关基因（CASP-3、-8、-9、Bax、Bcl-2、Bcl-XL、TP53、P21）的相对表达。

结果

hOPV感染以剂量和时间依赖性方式抑制HEp-2、A549和MCF-7细胞的生长。在感染复数（MOI）为5时，hOPV将A549细胞的活力降低至约16%，HEp-2细胞降低至22%，MCF-7细胞降低至28%（p = 0.001），而当细胞以MOI为1和2感染时未观察到明显的抑制作用。通过RT-PCR和IIF在感染的HEp-2、A549和MCF-7细胞中检测到hOPV mRNA和抗原。hOPV感染后，CASP-3、-8、-9以及Bax、TP53和p21 mRNA的表达增加，而抗凋亡基因Bcl-2、Bcl-xL的表达降低。在hOPV感染的A549细胞中，CASP-8和CASP-9、Bax、TP53和P21表达的增加倍数显著超过HEp-2或MCF-7细胞。